Gene therapy holds enormous potential for a new era in human medicine. These methodologies will allow treatment for conditions that heretofore have not been addressable by standard medical practice. One area that is especially promising is the ability to genetically engineer a cell to cause that cell to express a product not previously being produced in that cell, for example due to a mutation that inactivates the cognate gene in its genome. Examples of uses of this technology include the targeted correction of a disease-causing mutation, insertion of a gene encoding a novel therapeutic protein, insertion of a coding sequence encoding a protein that is lacking in the cell or in the individual, insertion of a wild type gene in a cell containing a mutated gene sequence, and insertion of a sequence that encodes a structural nucleic acid such as a microRNA or siRNA.
Transgenes can be delivered to a cell by a variety of ways, such that the transgene becomes integrated into the cell's own genome and is maintained there. In recent years, a strategy for transgene integration has been developed that uses cleavage with site-specific nucleases for targeted insertion into a chosen genomic locus (see, e.g., co-owned U.S. Pat. No. 7,888,121). Nucleases specific for targeted genes can be utilized such that the transgene construct is inserted by either homology directed repair (HDR) or by end capture during non-homologous end joining (NHEJ) driven processes. Targeted loci include “safe harbor” loci for example a CCR5 gene, a CXCR4 gene, a PPP1R12C (also known as AAVS1) gene, an albumin gene or a Rosa gene. See, e.g., U.S. Pat. Nos. 7,888,121; 7,972,854; 7,914,796; 7,951,925; 8,110,379; 8,409,861; 8,586,526; U.S. Patent Publications 20030232410; 20050208489; 20050026157; 20060063231; 20080159996; 201000218264; 20120017290; 20110265198; 20130137104; 20130122591; 20130177983 and 20130177960). Nuclease-mediated integration offers the prospect of improved transgene expression, increased safety and expressional durability, as compared to classic integration approaches that rely on random integration of the transgene, since it allows exact transgene positioning for a minimal risk of gene silencing or activation of nearby oncogenes. Nucleases include zinc finger nucleases (ZFN), transcription activator like effector nucleases (TALENs), mega or homing endonucleases, nuclease systems such as CRISPR/Cas that use a guide RNA to determine specificity, and fusions between nucleases such as mega-TALs.
Nuclease-mediated targeted integration can also be used for targeted gene correction of an endogenous locus. Gene correction can occur following nuclease cleavage as above by inserting a transgene of interest into the mutant endogenous locus. Alternatively, a mutant gene can be corrected by integrating a portion of the wild type or engineered sequence to replace (correct) the mutant portion of the gene locus. For gene correction, as with targeted integration of any exogenous sequence, sequence specific nucleases are used (e.g., ZFN, TALENs, CRISPR/Cas, meganucleases or megaTALs) to introduce a DSB in the endogenous gene of interest and a donor, typically comprising homology arms to the endogenous (mutant) gene, is integrated such that the endogenous (mutant) gene is altered by the donor. This approach can be used to correct mutations within an endogenous gene sequence and/or to insert engineered sequences for a desired purpose.
Red blood cells (RBCs), or erythrocytes, are the major cellular component of blood. In fact, RBCs account for one quarter of the cells in a human. Mature RBCs lack a nucleus and many other organelles in humans, and are full of hemoglobin, a metalloprotein found in RBCs that functions to carry oxygen to the tissues as well as carry carbon dioxide out of the tissues and back to the lungs for removal. The protein makes up approximately 97% of the dry weight of RBCs and it increases the oxygen carrying ability of blood by about seventy fold. Hemoglobin is a heterotetramer comprising two α-like globin chains and two β-like globin chains and 4 heme groups. In adults the α2β2 tetramer is referred to as Hemoglobin A (HbA) or adult hemoglobin. Typically, the alpha and beta globin chains are synthesized in an approximate 1:1 ratio and this ratio seems to be critical in terms of hemoglobin and RBC stabilization. In fact, in some cases where one type of globin gene is inadequately expressed (see below), reducing expression (e.g. using a specific siRNA) of the other type of globin, restoring this 1:1 ratio, alleviates some aspects of the mutant cellular phenotype (see Voon et at (2008) Haematologica 93(8):1288). In a developing fetus, a different form of hemoglobin, fetal hemoglobin (HbF) is produced which has a higher binding affinity for oxygen than Hemoglobin A such that oxygen can be delivered to the baby's system via the mother's blood stream. Fetal hemoglobin also contains two α globin chains, but in place of the adult β-globin chains, it has two fetal γ-globin chains (i.e., fetal hemoglobin is α2γ2). At approximately 30 weeks of gestation, the synthesis of γ globin in the fetus starts to drop while the production of β globin increases. By approximately 10 months of age after birth, the newborn's hemoglobin is nearly all α2β2 although some HbF persists into adulthood (approximately 1-3% of total hemoglobin). The regulation of the switch from production of γ to β is quite complex, and primarily involves an expressional down-regulation of γ globin with a simultaneous up-regulation of β globin expression.
Genetic defects in the sequences encoding the hemoglobin chains can be responsible for a number of diseases known as hemoglobinopathies, including sickle cell anemia and thalassemias. In the majority of patients with hemoglobinopathies, the genes encoding γ globin remain intact, but γ globin expression is relatively low due to normal gene repression occurring around parturition as described above.
Thalassemias are also diseases relating to hemoglobin and typically involve a reduced production of globin chains. This can occur through mutations in the regulatory regions of the genes or from a mutation in a globin coding sequence that results in reduced production. Alpha thalassemias are associated with people of Western Africa and South Asian descent, and may confer malarial resistance. Beta thalassemia is associated with people of Mediterranean descent, typically from Greece and the coastal areas of Turkey and Italy. Treatment of thalassemias usually involves blood transfusions and iron chelation therapy. Bone marrow transplants are also being used for treatment of people with severe thalassemias if an appropriate donor can be identified, but this procedure can have significant risks. Beta thalassemias are divided generally into three groups: i) Thalassemia trait or minor, where the patients are either carriers of a thalassemia disease allele or have very mild symptoms that may result in a mild anemia. ii) Thalassemia intermedia patients, where the lack of beta globin is great enough to cause a moderately severe anemia and significant health problems, including bone deformities and enlargement of the spleen. However, there is a wide range in the clinical severity of this condition, and the borderline between thalassemia intermedia and the most severe form, thalassemia major, can be confusing. The deciding factor seems to be the amount of blood transfusions required by the patient. iii) Thalassemia Major or Cooley's Anemia. This is the most severe form of beta thalassemia in which the complete lack of beta globin causes a life-threatening anemia that requires regular blood transfusions and extensive ongoing medical care. These extensive, lifelong blood transfusions lead to iron-overload which must be treated with chelation therapy to prevent early death from organ failure.
For beta thalessemias, in the late 1980s, it was estimated that approximately 54 mutations in the beta globin gene encompassed all known diseased beta globin genes; the number has since grown to over 200. The mutations are broken down into mutations which result in non-functional beta globin protein, including nonsense mutations and frameshift mutations; RNA processing mutations, including changes in the splice junctions and changes in splice consensus sequences as well as changes in internal intronic and exonic sequences that result in aberrant splicing; transcriptional mutants; polyA and RNA cleavage mutants; cap site mutants; and unstable mRNA mutants. These mutations result in either complete loss of beta globin mRNA in the cell (also referred to as “β−0”) or a reduced level of expression and mRNA accumulation (referred to as “β+”). (see, e.g., Kazazian and Boehm (1988) Blood vol 71 No 4: 1107). Patients with the milder β+ forms of beta thalessemia may have relatively normal lifespans, but those with the severe β−0 forms may die before age 30 if untreated, and if iron levels are not managed may also have a similarly shortened life span if given frequent transfusions. Of note, some specific mutations are more common than others; in particular, in some parts of Europe and the Middle East, a mutation known as “IVS1-1” (which stands for “intervening sequence 1, mutation number 1”) accounts for approximately 20% of all b-thalassemia major mutations. In this mutation, the “GT” dinucleotide at the beginning of intron 1 of the human beta-globin gene is changed to an “AT,” thereby disrupting a key signal (known as the “splice donor motif”) essential for accurate and efficient removal of the intron 1 from the beta-globin pre-mRNA. As a result, a significant reduction in beta-globin protein is observed during erythropoiesis, resulting in transfusion-dependent b-thalassemia major.
Thus, there remains a need for additional methods and compositions that can be used for genome editing, to correct an aberrant gene or alter the expression of others for example to treat beta thalassemias.